EP0845049A2 - Detection de genotypes du virus gb de l'hepatite - Google Patents

Detection de genotypes du virus gb de l'hepatite

Info

Publication number
EP0845049A2
EP0845049A2 EP96930516A EP96930516A EP0845049A2 EP 0845049 A2 EP0845049 A2 EP 0845049A2 EP 96930516 A EP96930516 A EP 96930516A EP 96930516 A EP96930516 A EP 96930516A EP 0845049 A2 EP0845049 A2 EP 0845049A2
Authority
EP
European Patent Office
Prior art keywords
sequence
hgbv
seq
oligonucleotide
dna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96930516A
Other languages
German (de)
English (en)
Inventor
Anthony S. Muerhoff
John N. Simons
Larry Birkenmeyer
Thomas P. Leary
James C. Erker
Suresh M. Desai
Isa K. Mushahwar
Michelle Chalmers
George J. Dawson
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Abbott Laboratories
Original Assignee
Abbott Laboratories
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Abbott Laboratories filed Critical Abbott Laboratories
Publication of EP0845049A2 publication Critical patent/EP0845049A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis
    • C12Q1/707—Specific hybridization probes for hepatitis non-A, non-B Hepatitis, excluding hepatitis D
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/318—Chemical structure of the backbone where the PO2 is completely replaced, e.g. MMI or formacetal
    • C12N2310/3181—Peptide nucleic acid, PNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays

Definitions

  • This invention relates generally to hepatitis GB virus and more particularly, relates to oligonucleotide primers and probes useful for detection of genotypes of hepatitis GB virus.
  • non-A, non-B (NANB) hepatitis-causing agent including multiple attacks of acute NANBH in intraveneous drug users, distinct incubation periods of patients acquiring NANBH post-transfusion, the outcome of cross-challenge chimpanzee experiments, the ultrastructural liver pathology of infected chimpanzees and the differential resistance of the putative agents to chloroform.
  • NANBH non-A, non-B
  • HCV hepatitis C virus
  • genotypes vary in nucleotide and amino acid sequence as well as in severity of the disease and geographical location. See, for example, G. Dawson et al., "Recent Developments in the Molecular Biology of the Hepatitis Virus," in Current Hepatology. G. Gitnick, ed., Mosby Publishers (1995, in press). Thus, detection of genotypes of HGBV can aid in the clinical and epidemiological understanding ofthe virus.
  • HGBV detection in test samples can be enhanced by the use of DNA hybridization assays which utilize DNA oligomers as hybridization probes. Since the amount of DNA target nucleotides present in a test sample may be in minute amounts, target DNA usually is amplified and then detected. Methods for amplifying and detecting a target nucleic acid sequence that may be present in a test sample are well-known in the art. Such methods include the polymerase chain reaction (PCR) described in U.S. Patents 4,683,195 and 4,683,202 which are inco ⁇ orated herein by reference, the ligase chain reaction (LCR) described in EP- A-320 308 , gap LCR (GLCR) described in European Patent Application EP-A- 439 182 and U.S.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • LCR ligase chain reaction
  • GLCR gap LCR
  • Patent No. 5,427,930 which are inco ⁇ orated herein by reference, multiplex LCR described in International Patent Application No. WO 93/20227, NASBA and the like. These methods have found widespread application in the medical diagnostic field as well as in the fields of genetics, molecular biology and biochemistry.
  • DNA probes derived from HGBV which can detect HGBV in test samples of individuals suspected of being infected with HGBV and test kits which utilize these probes.
  • Such probes could greatly enhance the ability ofthe medical community to more accurately diagnose acute and/or chronic viral hepatitis and could provide a safer blood and organ supply by detecting non-A, non-B and non-C hepatitis in these blood and organ donations, and could provide a better understanding of the prevalence of HGBV in the population, epidemiology of the disease caused by HGBV and the prognosis of infected individuals.
  • the present invention provides unique primers for HGBV-C detection.
  • primers are identified as SEQUENCE ID NO 51, SEQUENCE ID NO 53, SEQUENCE ID NO 54, SEQUENCE ID NO 55, SEQUENCE ID NO 56, SEQUENCE ID NO 57, and SEQUENCE ID NO 87, and complements thereof.
  • the primer(s) disclosed herein can detect the presence of HGBV-C and are not reactive with HGBV-A or HGBV-B.
  • the present invention also provides a method of detection target HGBV-C nucleotides in a test sample, comprising contacting a target HGBV nucleotide with at least one oligonucleotide and detecting the presence of the target in the test sample.
  • the oiigonucleotides can be selected from the group consisting of SEQUENCE ID NO 51 i SEQUENCE ID NO 53, SEQUENCE ID NO 54,
  • SEQUENCE ID NO 55 SEQUENCE ID NO 56, SEQUENCE ID NO 57, and SEQUENCE ID NO 87, and complements thereof.
  • the oiigonucleotides utilized also can be selected from the group consisting of SEQUENCE ID NO 2, SEQUENCE ID NO 3, SEQUENCE ID NO 4, SEQUENCE ID NO 5, SEQUENCE ID NO 6, SEQUENCE ID NO 9, SEQUENCE ID NO 10, SEQUENCE ID NO 11, SEQUENCE ID NO 13, SEQUENCE ID NO 13, SEQUENCE ID NO 14, SEQUENCE ID NO 15, SEQUENCE ID NO 16, SEQUENCE ID NO 17, SEQUENCE ID NO 18, SEQUENCE ID NO 19, SEQUENCE ID NO 20, SEQUENCE ID NO 21, SEQUENCE ID NO 22, SEQUENCE ID NO 23, SEQUENCE ID NO 24, SEQUENCE ID NO 60, SEQUENCE ID NO 61, SEQUENCE ID NO 62, SEQUENCE ID NO 63, SEQUENCE ID NO 64, SEQUENCE ID NO 65, SEQUENCE ID NO 66, SEQUENCEIDNO67,SEQUENCEIDNO68,SEQU
  • the present invention also provides a method of amplifying 5' end cDNA of hepatitis GB-C (HGBV-C) virus in a test sample, comprising performing reverse transcription with random primers and test sample, amplifying the cDNA so obtained by using other oligonucleotide primers as sense and antisense primers in a first stage PCR to obtain amplified cDNA of HGBV-C, and detecting the presence of the amplicon (amplified cDNA) in the test sample.
  • At least one oligonucleotide used as a sense primer can be selected from the group consisting of SEQUENCE ID NO. 51, SEQUENCE ID NO 53 andSEQUENCE ID NO 56.
  • At least one oligonucleotide used as an antisense primer can be selected from the group consisting of SEQUENCE ID NO 54, SEQUENCE ID NO 55,SEQUENCE ID NO 57 and SEQUENCE ID NO 87.
  • the present invention also provides a method for detecting target hepatitis GB-C virus (HGBV-C) in a test sample suspected of containing target HGBV-C, comprising contacting the test sample with at least one oligonucleotide of HGBV-C as a sense primer and at least one oligonucleotide of HGBV-C as an anti-sense primer and amplifying same to obtain a first stage reaction product; then contacting the first stage reaction product with at least one ofthe oiigonucleotides used previously and a second oligonucleotide, with the proviso that the second oligonucleotide is located 3' to the first oligonucleotide utilized and is of opposite sense to the first oligonucleotide, and then detecting the HGBV target.
  • HGBV-C target hepatitis GB-C virus
  • the first stage PCR reaction can comprise utilizing at least one oligonucleotide selected from the group consisting of SEQUENCE ID NO 51, SEQUENCE ID NO 56 and SEQUENCE ID NO 53 as a sense primer and utilizing at least one oligonucleotide selected from the group consisting of SEQUENCE ID NO 54, SEQUENCE ID NO 55, SEQUENCE ID NO 57 and SEQUENCE ID NO 87 as an anti-sense primer.
  • the products of this first stage PCR then can be further amplified in a second stage PCR reaction which comprises utilizing at least one oligonucleotide selected from the group consisting of SEQUENCE ID NO 56 and SEQUENCE ID NO 53 as a sense primer, and utilizing at least one oligonucleotide selected from the group consisting of SEQUENCE ID NO 54, SEQUENCE ID NO 55 and SEQUENCE ID NO 57 as an anti-sense primer, with the proviso that the second oligonucleotide is located 3' to the first oligonucleotide utilized and is of opposite sense to the first oligonucleotide.
  • the amplification in all methods can be performed by the polymerase chain reaction (PCR).
  • the test sample in these methods can be attached to a solid phase prior to performing the methods steps outlined hereinabove.
  • the detection step of these methods can comprise utilizing a detectable measureable signal generating compound Oabel) which generates a measurable signal.
  • the label can be attached to a solid phase.
  • GAP LCR also can be performed according to the invention, utilizing
  • SEQUENCE ID NO 35 SEQUENCE ID NO 36, SEQUENCE ID NO 37, SEQUENCE ID NO 38, SEQUENCE ID NO 39, SEQUENCE ID NO 40, SEQUENCE ID NO 41, SEQUENCE ID NO 42, SEQUENCE ID NO 43, SEQUENCE ID NO 44, SEQUENCE ID NO 45, SEQUENCE ID NO 46, SEQUENCE ID NO 47, SEQUENCE ID NO 48, SEQUENCE ID NO 49 and SEQUENCE ID NO 50, for genotype differentiation.
  • FIGURES 1 A through FIGURE IF show the nucleotide alignment of the HGBV-C isolates.
  • FIGURE 2 shows a phylogenetic tree of the genotypes of HGBV-C.
  • the present invention provides characterization of a newly ascertained genotypes of etiological agents of non- A, non-B, non-C, non-D and non-E hepatitis-causing agents, collectively so-termed "Hepatitis GB Virus," or "HGBV.”
  • the present invention provides a method for detecting HGBV genotypes, oiigonucleotides useful for detecting HGBV and oiigonucleotides useful for differentiating HGBV-C genotypes.
  • kits containing reagents which can be used for the detection of HGBV genotypes such reagents comprising a polynucleotide probe containing a nucleotide sequence from HGBV of about 8 or more nucleotides in a suitable container
  • Hepatitis GB Virus or "HGBV”, as used herein, collectively denotes a viral species which causes non-A, non-B, non-C, non-D, non-E hepatitis in man, and attenuated strains or defective interfering particles derived therefrom. This may include acute viral hepatitis transmitted by contaminated foodstuffs, drinking water, and the like; hepatitis due to HGBV transmitted via person to person contact (including sexual transmission, respiratory and parenteral routes) or via intraveneous drug use. The methods as described herein will allow the identification of individuals who have acquired HGBV.
  • the HGBV isolates are specifically referred to as "HGB V-A”, “HGB V-B “ and "HGBV-C.”
  • the HGBV genome is comprised of RNA. Analysis of the nucleotide sequence and deduced amino acid sequence of the HGBV reveals that viruses of this group have a genome organization similar to that of the Flaviridae family. Based primarily, but not exclusively, upon similarities in genome organization, the International Committee on the Taxonomy of Viruses has recommended that this family be composed of three genera:
  • Flavivirus, Pestivirus, and the hepatitis C group Similarity searches at the amino acid level reveal that the hepatitis GB virus subclones have some, albeit low, sequence resemblance to hepatitis C virus. It now has been demonstrated that HGBV-C is not a genotype of HCV. See, for example, U.S. Serial No. 08/417,629, filed April 6, 1995, previously inco ⁇ orated herein by reference.
  • the term "similarity” and/or "identity” are used herein to describe the degree of relatedness between two polynucleotides or polypeptide sequences.
  • amino acid sequence “similarity” and/or “identity” are well-known in the art and include, for example, directly determining the amino acid sequence and comparing it to the sequences provided herein; determining the nucleotide sequence of the genomic material of the putative HGBV (usually via a cDNA intermediate), and determining the amino acid sequence encoded therein, and comparing the corresponding regions.
  • identity is meant the exact match-up of either the nucleotide sequence of HGBV and that of another strain(s) or the amino acid sequence of HGBV and that of another strain(s) at the appropriate place on each genome.
  • similarity is meant the exact match-up of amino acid sequence of HGBV and that of another strain(s) at the appropriate place, where the amino acids are identical or possess similar chemical and/or physical properties such as charge or hydrophobicity.
  • GAP program are capable of calculating both the identity and similarity between two polynucleotide or two polypeptide sequences.
  • Other programs for calculating identity and similarity between two sequences are known in the art.
  • HGBV-A HGBV-A
  • HGBV-B HGBV-C
  • HGBV-C HGBV-C
  • the overall nucleotide sequence identity of the genomes between HGBV-A, HGBV-B or HGBV-C and a strain of one of these hepatitis GB viruses will be about 45% or greater, since it is now believed that the HGBV strains may be genetically related, preferably about 60% or greater, and more preferably, about 80% or greater.
  • the overall sequence identity of the genomes between HGBV-A and a strain of HGBV-A at the amino acid level will be about 35% or greater since it is now believed that the HGBV strains may be genetically related, preferably about 40% or greater, more preferably, about 60% or greater, and even more preferably, about 80% or greater.
  • the HGBV strains may be genetically related, preferably about 40% or greater, more preferably, about 60% or greater, and even more preferably, about 80% or greater.
  • the overall sequence identity of the genomes between HGBV-B and a strain of HGBV-B at the amino acid level will be about 35% or greater since it is now believed that the HGBV strains may be genetically related, preferably about 40% or greater, more preferably, about 60% or greater, and even more preferably, about 80% or greater.
  • the HGBV strains may be genetically related, preferably about 40% or greater, more preferably, about 60% or greater, and even more preferably, about 80% or greater.
  • the overall sequence identity of the genomes between HGBV-C and a strain of HGBV-C at the amino acid level will be about 35% or greater since it is now believed that the HGBV strains may be genetically related, preferably about 40% or greater, more preferably, about 60% or greater, and even more preferably, about 80% or greater.
  • the HGBV strains may be genetically related, preferably about 40% or greater, more preferably, about 60% or greater, and even more preferably, about 80% or greater.
  • a polynucleotide "derived from" a designated sequence for example, the HGBV cDNA, or from the HGBV genome refers to a polynucleotide sequence which is comprised of a sequence of approximately at least about 6 nucleotides, is preferably at least about 8 nucleotides, is more preferably at least about 10-12 nucleotides, and even more preferably is at least about 15-20 nucleotides corresponding, i.e., similar to or complementary to, a region of the designated nucleotide sequence.
  • the sequence of the region from which the polynucleotide is derived is similar to or complementary to a sequence which is unique to the HGBV genome.
  • Whether or not a sequence is complementary to or similar to a sequence which is unique to an HGBV genome can be determined by techniques known to those skilled in the art. Comparisons to sequences in databanks, for example, can be used as a method to determine the uniqueness of a designated sequence. Regions from which sequences may be derived include but are not limited to regions encoding specific epitopes, as well as non-translated and/or non-transcribed regions.
  • the derived polynucleotide will not necessarily be derived physically from the nucleotide sequence of HGBV, but may be generated in any manner, including but not limited to chemical synthesis, replication or reverse transcription or transcription, which are based on the information provided by the sequence of bases in the region(s) from which the polynucleotide is derived.
  • combinations of regions corresponding to that of the designated sequence may be modified in ways known in the art to be consistent with an intended use.
  • polynucleotide as used herein means a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, the term includes double- and single-stranded DNA, as well as double- and single-stranded RNA. It also includes modifications, either by methylation and/or by capping, and unmodified forms of the polynucleotide.
  • HGBV containing a sequence corresponding to a cDNA means that the
  • HGBV contains a polynucleotide sequence which is similar to or complementary to a sequence in the designated DNA.
  • the degree of similarity or complementarity to the cDNA will be approximately 50% or greater, will preferably be at least about 70%, and even more preferably will be at least about 90%.
  • the sequence which corresponds will be at least about 70 nucleotides, preferably at least about 80 nucleotides, and even more preferably at least about 90 nucleotides in length.
  • the correspondence between the HGBV and the cDNA can be determined by methods known in the art, and include, for example, a direct comparison of the sequenced material with the cDNAs described, or hybridization and digestion with single strand nucleases, followed by size determination of the digested fragments.
  • Purified viral polynucleotide refers to an HGBV genome or fragment thereof which is essentially free, i.e., contains less than about 50%, preferably less than about 70%, and even more preferably, less than about 90% of polypeptides with which the viral polynucleotide is naturally associated.
  • Techniques for purifying viral polynucleotides include, for example, disruption of the particle with a chaotropic agent, and separation of the polynucleotide(s) and polypeptides by ion-exchange chromatography, affinity chromatography, and sedimentation according to density.
  • purified viral polypeptide means an HGBV polypeptide or fragment thereof which is essentially free, that is, contains less than about 50%, preferably less than about 70%, and even more preferably, less than about 90% of cellular components with which the viral polypeptide is naturally associated. Methods for purifying are known to the routineer.
  • Polypeptide indicates a molecular chain of amino acids and does not refer to a specific length of the product. Thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term, however, is not intended to refer to post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like.
  • polypeptide or "amino acid sequence derived from a designated nucleic acid sequence or from the HGBV genome refers to a polypeptide having an amino acid sequence identical to that of a polypeptide encoded in the sequence or a portion thereof wherein the portion consists of at least 3 to 5 amino acids, and more preferably at least 8 to 10 amino acids, and even more preferably 15 to 20 amino acids, or which is immunologically identifiable with a polypeptide encoded in the sequence.
  • a "recombinant polypeptide” as used herein means at least a polypeptide of genomic, semisynthetic or synthetic origin which by virtue of its origin or manipulation is not associated with all or a portion of the polypeptide with which it is associated in nature or in the form of a library andor is linked to a polynucleotide other than that to which it is linked in nature.
  • a recombinant or derived polypeptide is not necessarily translated from a designated nucleic acid sequence of HGBV or from an HGBV genome. It also may be generated in any manner, including chemical synthesis or expression of a recombinant expression system, or isolation from mutated HGBV.
  • synthetic peptide as used herein means a polymeric form of amino acids of any length, which may be chemically synthesized by methods well- known to the routineer. These synthetic peptides are useful in various applications.
  • Recombinant host cells refer to cells which can be, or have been, used as recipients for recombinant vector or other transfer DNA, and include the original progeny of the original cell which has been transfected.
  • replicon means any genetic element, such as a plasmid, a chromosome or a virus, that behaves as an autonomous unit of polynucleotide replication within a cell. That is, it is capable of replication under its own control.
  • a “vector” is a replicon in which another polynucleotide segment is attached, such as to bring about the replication and/or expression of the attached segment.
  • control sequence refers to polynucleotide sequences which are necessary to effect the expression of coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism. In prokaryotes, such control sequences generally include promoter, ribosomal binding site and terminators; in eukaryotes, such control sequences generally include promoters, terminators and, in some instances, enhancers.
  • control sequence thus is intended to include at a minimum all components whose presence is necessary for expression, and also may include additional components whose presence is advantageous, for example, leader sequences.
  • operably linked refers to a situation wherein the components described are in a relationship permitting them to function in their intended manner.
  • a control sequence operably linked to a coding sequence is ligated in such a manner that expression of the coding sequence is achieved under conditions compatible with the control sequences.
  • ORF open reading frame
  • ORF refers to a region of a polynucleotide sequence which encodes a polypeptide; this region may represent a portion of a coding sequence or a total coding sequence.
  • a “coding sequence” is a polynucleotide sequence which is transcribed into mRNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5" -terminus and a translation stop codon at the 3' -terminus.
  • a coding sequence can include, but is not limited to, mRNA, cDNA, and recombinant polynucleotide sequences.
  • the term "immunologically identifiable with/as” refers to the presence of epitope(s) and polypeptide(s) which also are present in and are unique to the designated polypeptide(s), usually HGBV proteins.
  • Immunological identity may be determined by antibody binding and/or competition in binding. These techniques are known to the routineer and also are described herein.
  • the uniqueness of an epitope also can be determined by computer searches of known data banks, such as GenBank, for the polynucleotide sequences which encode the epitope, and by amino acid sequence comparisons with other known proteins.
  • epitope means an antigenic determinant of a polypeptide.
  • an epitope can comprise three amino acids in a spatial conformation which is unique to the epitope. Generally, an epitope consists of at least five such amino acids, and more usually, it consists of at least eight to ten amino acids. Methods of examining spatial conformation are known in the art and include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance.
  • the term "individual” as used herein refers to vertebrates, particularly members of the mammalian species and includes but is not limited to domestic animals, sports animals, primates and humans; more particularly the term refers to tamarins and humans.
  • a polypeptide is "immunologically reactive" with an antibody when it binds to an antibody due to antibody recognition of a specific epitope contained within the polypeptide. Immunological reactivity may be determined by antibody binding, more particularly by the kinetics of antibody binding, and/or by competition in binding using as competitor(s) a known polypeptide(s) containing an epitope against which the antibody is directed. The methods for determining whether a polypeptide is immunologically reactive with an antibody are known in the art.
  • immunogenic polypeptide containing an HGBV epitope means naturally occurring HGBV polypeptides or fragments thereof, as well as polypeptides prepared by other means, for example, chemical synthesis or the expression of the polypeptide in a recombinant organism.
  • transformation refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion. For example, direct uptake, transduction, or f-mating are included.
  • the exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome.
  • Treatment refers to prophylaxis and/or therapy.
  • the term “plus strand” (or “+”) as used herein denotes a nucleic acid that contains the sequence that encodes the polypeptide. '
  • minus strand denotes a nucleic acid that contains a sequence that is complementary to that of the "plus” strand.
  • “Positive stranded genome” of a virus denotes that the genome, whether RNA or DNA, is single-stranded and encodes a viral polypeptide(s).
  • the term "test sample” refers to a component of an individual's body which is the source of the analyte (such as, antibodies of interest or antigens of interest). These components are well known in the art.
  • test samples include biological samples which can be tested by the methods of the present invention described herein and include human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external secretions of the respiratory, intestinal and genitorurinary tracts, tears, saliva, milk, white blood cells, myelomas and the like; biological fluids such as cell culture supematants; fixed tissue specimens; and fixed cell specimens.
  • human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external secretions of the respiratory, intestinal and genitorurinary tracts, tears, saliva, milk, white blood cells, myelomas and the like
  • biological fluids such as cell culture supematants
  • fixed tissue specimens fixed cell specimens.
  • Purified HGBV refers to a preparation of HGBV which has been isolated from the cellular constituents with which the virus is normally associated, and from other types of viruses which may be present in the infected tissue.
  • the techniques for isolating viruses are known to those skilled in the art and include, for example, centrifugation and affinity chromatography.
  • PNA denotes a "peptide nucleic analog" which may be utilized in a procedure such as an assay described herein to determine the presence of a target.
  • PNAs are neutrally charged moieties which can be directed against RNA targets or DNA.
  • PNA probes used in assays in place of, for example, the DNA probes of the present invention offer advantages not achievable when DNA probes are used. These advantages include manufacturability, large scale labeling, reproducibility, stability, insensitivity to changes in ionic strength and resistance to enzymatic degradation which is present in methods utilizing DNA or RNA.
  • These PNAs can be labeled with such signal generating compounds as fluorescein, radionucleotides, chemiluminescent compounds, and the like.
  • PNAs or other nucleic acid analogs such as mo ⁇ holino compounds thus can be used in assay methods in place of DNA or RNA.
  • assays are described herein utilizing DNA probes, it is within the scope of the routineer that PNAs or mo ⁇ holino compounds can be substituted for RNA or DNA with appropriate changes if and as needed in assay reagents.
  • Solid phases are known to those in the art and include the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, nitrocellulose strips, membranes, microparticles such as latex particles, sheep (or other animal) red blood cells, duracytes and others.
  • the "solid phase” is not critical and can be selected by one skilled in the art.
  • latex particles, microparticles, magnetic or non-magnetic beads, membranes, plastic tubes, walls of microtiter wells, glass or silicon chips, sheep (or other suitable animal's) red blood cells and duracytes are all suitable examples.
  • Suitable methods for immobilizing probes on solid phases include ionic, hydrophobic, covalent interactions and the like.
  • a “solid phase”, as used herein, refers to any material which is insoluble, or can be made insoluble by a subsequent reaction.
  • the solid phase can be chosen for its intrinsic ability to attract and immobilize the capture reagent.
  • the solid phase can retain an additional receptor which has the ability to attract and immobilize the capture reagent.
  • the additional receptor can include a charged substance that is oppositely charged with respect to the capture reagent itself or to a charged substance conjugated to the capture reagent.
  • the receptor molecule can be any specific binding member which is immobilized upon (attached to) the solid phase and which has the ability to immobilize the capture reagent through a specific binding reaction.
  • the receptor molecule enables the indirect binding of the capture reagent to a solid phase material before the performance of the assay or during the performance of the assay.
  • the solid phase thus can be a plastic, derivatized plastic, magnetic or non ⁇ magnetic metal, glass or silicon surface of a test tube, microtiter well, sheet, bead, microparticle, chip, sheep (or other suitable animal's) red blood cells, duracytes and other configurations known to those of ordinary skill in the art. It is contemplated and within the scope of the invention that the solid phase also can comprise any suitable porous material with sufficient porosity to allow access by detection antibodies and a suitable surface affinity to bind antigens. Microporous structures are generally preferred, but materials with gel structure in the hydrated state may be used as well.
  • Such useful solid supports include but are not limited to natural polymeric carbohydrates and their synthetically modified, cross-linked or substituted derivatives, such as agar, agarose, cross-linked alginic acid, substituted and cross-linked guar gums, cellulose esters, especially with nitric acid and carboxylic acids, mixed cellulose esters, and cellulose ethers; natural polymers containing nitrogen; synthetic polymers which may be prepared with suitably porous structures, such as vinyl polymers; porous inorganic materials such as sulfates or carbonates of alkaline earth metals and magnesium, including barium sulfate, calcium sulfate, calcium carbonate, silicates of alkali and alkaline earth metals, aluminum and magnesium; and aluminum or silicon oxides or hydrates, such as clays, alumina, talc, kaolin, zeolite, silica gel, or glass (these materials may be used as filters with the above polymeric materials); and mixtures or copolymers of the above classes, such as
  • porous structure of nitrocellulose has excellent abso ⁇ tion and adso ⁇ tion qualities for a wide variety of reagents.
  • Nylon also possesses similar characteristics and also is suitable.
  • porous solid supports described hereinabove are preferably in the form of sheets of thickness from about 0.01 to 0.5 mm, preferably about 0.1 mm.
  • the pore size may vary within wide limits, and is preferably from about 0.025 to 15 microns, especially from about 0.15 to 15 microns.
  • the surfaces of such supports may be activated by chemical processes which cause covalent linkage of the antigen or antibody to the support. The irreversible binding of the antigen or antibody is obtained, however, in general, by adso ⁇ tion on the porous material by poorly understood hydrophobic forces.
  • Suitable solid supports also are described in U.S. Patent Application Serial No.227,272.
  • the “indicator reagent” “comprises a “signal generating compound” (also termed a “label”) which is capable of generating and generates a measurable signal detectable by external means conjugated (attached) to a specific binding member for HGBV.
  • “Specific binding member” as used herein means a member of a specific binding pair. That is, two different molecules where one of the molecules through chemical or physical means specifically binds to the second molecule.
  • the indicator reagent also can be a member of any specific binding pair, including either hapten-anti-hapten systems such as biotin or anti-biotin, avidin or biotin, a carbohydrate or a lectin, a complementary nucleotide sequence, an effector or a receptor molecule, an enzyme cofactor and an enzyme, an enzyme inhibitor or an enzyme, and the like.
  • specific binding pairs can include members that are analogs of the original specific binding members, for example, an analyte- analog.
  • An immunoreactive specific binding member can be an antibody or fragment thereof, an antigen or fragment thereof, or an antibody/antigen complex including those formed by recombinant DNA molecules that is capable of binding either to HGBV as in a sandwich assay, to the capture reagent as in a competitive assay, or to the ancillary specific binding member as in an indirect assay.
  • labels include chromogens, catalysts such as enzymes, luminescent compounds such as fluorescein and rhodamine, chemiluminescent compounds such as dioxetanes, acridiniums, phenanthridiniums and luminol,- radioactive elements, and direct visual labels.
  • luminescent compounds such as fluorescein and rhodamine
  • chemiluminescent compounds such as dioxetanes, acridiniums, phenanthridiniums and luminol
  • - radioactive elements and direct visual labels.
  • enzymes include alkaline phosphatase, horseradish peroxidase, beta-galactosidase, and the like.
  • the selection of a particular label is not critical, but it will be capable of producing a signal either by itself or in conjunction with one or more additional substances.
  • a label can be directly detectable, as with, for example, radioisotopes, fluorophores, chemiluminophores, enzymes, colloidal particles, fluorescent microparticles and the like; or a label may be indirectly detectable, as with, for example, specific binding members.
  • direct labels may require additional components such as but not limited to substrates, triggering reagents, light, and the like to enable detection of the label.
  • indirect labels are typically used in combination with a conjugate.
  • a "conjugate” is typically a specific binding member which has been attached or coupled to a directly detectable label. Coupling chemistries for synthesizing a conjugate are well known in the art and can include, for example, any chemical means and/or physical means that does not destroy the specific binding property of the specific binding member or the detectable property of the label.
  • hapten refers to a partial antigen or non-protein binding member which is capable of binding to an antibody, but which is not capable of eliciting antibody formation unless coupled to a carrier protein.
  • haptens include biotin, avidin, adamantine and carbazole.
  • analyte is the substance to be detected which may be present in the test sample.
  • the analyte can be any substance for which there exists a naturally occurring specific binding member (such as, an antibody), or for which a specific binding member can be prepared.
  • an analyte is a substance that can bind to one or more specific binding members in an assay.
  • “Analyte” also includes any antigenic substances such as target nucleotide sequences, haptens, antibodies, and combinations thereof.
  • the analyte can be detected by means of naturally occurring specific binding partners (pairs) such as the use of intrinsic factor protein as a member of a specific binding pair for the determination of Vitamin B 12, the use of folate-binding protein to determine folic acid, or the use of a lectin as a member of a specific binding pair for the determination of a carbohydrate.
  • the analyte can include a protein, a peptide, an amino acid, a nucleotide target of RNA or DNA or of PNA, and the like.
  • the methods of the present invention can be adapted for use in systems which utilize microparticle technology including in automated and semi- automated systems wherein the solid phase comprises a microparticle (magnetic or non-magnetic).
  • Such systems include those described in pending U. S. Patent Applications 425,651 and 425,643, which co ⁇ espond to published EPO applications Nos. EP 0425 633 and EP 0424634, respectively.
  • SPM scanning probe microscopy
  • the HGBV group of viruses may be detectable in assays by use of a synthetic, recombinant or native probe that is common to all HGBV viruses. It also is within the scope of the present invention that different synthetic, recombinant or native probes identifying different epitopes from HGBV-A, HGBV-B, HGBV-C, or yet other HGBV viruses, can be used in assay formats. In the later case, these can be coated onto one solid phase, or each separate probe may be coated on separate solid phases, such as microparticles, and then combined to form a mixture of probes which can be later used in assays. Such variations of assay formats are known to those of ordinary skill in the art and are discussed hereinbelow.
  • the reagents and methods of the present invention are made possible by the provision of a family of closely related nucleotide sequences present in the plasma, serum or liver homogenate of an HGBV infected individual, either tamarin or human.
  • This family of nucleotide sequences is not of human or tamarin origin, since it hybridizes to neither human nor tamarin genomic DNA from uninfected individuals, since nucleotides of this family of sequences are present only in liver (or liver homogenates), plasma or serum of individuals infected with HGBV, and since the sequence is not present in GenBank®.
  • the family of sequences will show no significant identity at the nucleic acid level to sequences contained within the HAV, HBV, HCV, HDV and HEV genome, and low level identity, considered not significant, as translation products.
  • Infectious sera, plasma or liver homogenates from HGBV infected humans contain these polynucleotide sequences, whereas sera, plasma or liver homogenates from non- infected humans do not contain these sequences.
  • Northern blot analysis of infected liver with some of these polynucleotide sequences demonstrate that they are derived from a large RNA transcript similar in size to a viral genome.
  • Sera, plasma or liver homogenates from HGB V-infected humans contain antibodies which bind to this polypeptide, whereas sera, plasma or liver homogenates from non-infected humans do not contain antibodies to this polypeptide; these antibodies are induced in individuals following acute non- A, non-B, non-C, non-D and non- E hepatitis infection.
  • the sequence is a viral sequence, wherein the virus causes or is associated with non- A, non-B, non-C, non-D and non-E hepatitis.
  • DNA probes and polypeptides useful in diagnosing non- A, non-B, non-C, non-D, non-E hepatitis due to HGBV infections, and in screening blood donors, donated blood, blood products and individuals for infection.
  • DNA oligomers of about eight to ten nucleotides, or larger, which are useful as hybridization probes or PCR primers to detect the presence of the viral genome in, for example, sera of subjects suspected of harboring the virus, or for screening donated blood for the presence of the virus.
  • nucleic acid sequences also allows the design and production of HGBV specific polypeptides which are useful as diagnostic reagents for the presence of antibodies raised during infection with HGBV.
  • Antibodies to purified polypeptides derived from the nucleic acid sequences may also be used to detect viral antigens in infected individuals and in blood.
  • These nucleic acid sequences also enable the design and production of polypeptides which may be used as vaccines against HGBV, and also for the production of antibodies, which then may be used for protection of the disease, and/or for therapy of HGBV infected individuals.
  • oligomers of approximately eight nucleotides or more can be prepared, either by excision or synthetically, which hybridize with the HGBV genome and are useful in identification of the viral agent(s), further characterization of the viral genome, as well as in detection of the virus(es) in diseased individuals.
  • the natural or derived probes for HGBV polynucleotides are a length which allows the detection of unique viral sequences by hybridization. While six to eight nucleotides may be a workable length, sequences of ten to twelve nucleotides are prefe ⁇ ed, and those of about 20 nucleotides may be most prefe ⁇ ed.
  • probes can be prepared using routine, standard methods including automated oligonucleotide synthetic methods. A complement of any unique portion of the HGBV genome will be satisfactory. Complete complementarity is desirable for use as probes, although it may be unnecessary as the length of the fragment is increased.
  • the test sample to be analyzed such as blood or serum
  • the test sample to be analyzed may be treated such as to extract the nucleic acids contained therein.
  • the resulting nucleic acid from the sample may be subjected to gel electrophoresis or other size separation techniques; or, the nucleic acid sample may be dot-blotted without size separation.
  • the probes then are labeled.
  • Suitable labels and methods for attaching labels to probes are known in the art, and include but are not limited to radioactive labels inco ⁇ orated by nick translation or kinasing, biotin, fluorescent and chemiluminescent probes. Examples of many of these labels are disclosed herein.
  • the nucleic acids extracted from the sample then are treated with the labeled probe under hybridization conditions of suitable stringencies.
  • the probes can be made completely complementary to the HGBV genome. Therefore, usually high stringency conditions are desirable in order to prevent false positives. However, conditions of high stringency should be used only if the probes are complementary to regions of the HGBV genome which lack heterogeneity.
  • the stringency of hybridization is determined by a number of factors during the washing procedure, including temperature, ionic strength, length of time and concentration of formamide. See, for example, J. Sambrook (supra).
  • Hybridization can be carried out by a number of various techniques. Amplification can be performed, for example, by Ligase Chain Reaction (LCR), Polymerase Chain Reaction (PCR), Q-beta replicase, NASBA, etc.
  • the HGBV genome sequences may be present in serum of infected individuals at relatively low levels, for example, approximately 10-*-- 10- sequences per ml. This level may require that amplification techniques such as the LCR or the PCR be used in hybridization assays. Such techniques are known in the art.
  • the "Bio-Bridge" system uses terminal deoxynucleotide transferase to add unmodified 3'-poly-dT-tails to a nucleic acid probe (Enzo Biochem. Co ⁇ .). The poly dt-tailed probe is hybridized to the target nucleotide sequence, and then to a biotin-modified poly-A.
  • EP 124221 there is described a DNA hybridization assay wherein the analyte is annealed to a single-stranded DNA probe that is complementary to an enzyme-labeled oligonucleotide, and the resulting tailed duplex is hybridized to an enzyme-labeled oligonucleotide.
  • EP 204510 describes a DNA hybridization assay in which analyte DNA is contacted with a probe that has a tail, such as a poly-dT-tail, an amplifier strand that has a sequence that hybridizes to the tail ofthe probe, such as a poly-A sequence, and which is capable of binding a plurality of labeled strands.
  • the technique first may involve amplification ofthe target HGBV sequences in sera to approximately 10 ⁇ sequences/ml. This may be accomplished by following the methods described by Saiki et al., Nature 324: 163 (1986).
  • the amplified sequence(s) then may be detected using a hybridization assay such as those known in the art.
  • the probes can be packaged in diagnostic kits which include the probe nucleic acid sequence which sequence may be labeled; alternatively, the probe may be unlabeled and the ingredients for labeling could be included with the kit.
  • the kit also may contain other suitably packaged reagents and materials needed or desirable for the particular hybridization protocol, for example, standards as well as instructions for performing the assay.
  • Fluorescence in situ hybridization also can be performed utilizing the reagents described herein.
  • In situ hybridization involves taking mo ⁇ hologically intact tissues, cells or chromosomes through the nucleic acid hybridization process to demonstrate the presence of a particular piece of genetic information and its specific location within individual cells. Since it does not require homogenization of cells and extraction ofthe target sequence, it provides precise localization and distribution of a sequence in cell populations. In situ hybridization can identify the sequence of interest concentrated in the cells containing it. It also can identify the type and fraction of the cells in a heterogeneous cell population containing the sequence of interest. DNA and RNA can be detected with the same assay reagents.
  • PNAs or mo ⁇ holino compounds can be utilized in FISH methods to detect targets without the need for amplification. If increased signal is desired, multiple fluorophores can be used to increase signal and thus, sensitivity of the method.
  • Various methods of FISH are known, including a one-step method using multiple oiigonucleotides or the conventional multi-step method. It is within the scope of the present invention that these types of methods can be automated by various means including flow cytometry and image analysis.
  • Assays as described herein may utilize one viral antigen derived from any clone-containing HGBV nucleic acid sequence, or from the composite nucleic acid sequences derived from the HGBV nucleic acid sequences in these clones, or from the HGBV genome from which the nucleic acid sequences in these clones are derived.
  • the assay may use a combination of viral antigens derived from these sources. It also may use, for example, a monoclonal antibody directed against the same viral antigen, or polyclonal antibodies directed against different viral antigens.
  • Assays can include but are not limited to those based on competition, direct reaction or sandwich-type assays.
  • Assays may use solid phases or may be performed by immunoprecipitation or any other methods which do not utilize solid phases. Examples of assays which utilize labels as the signal generating compound and those labels are described herein. Signals also may be amplified by using biotin and avidin, enzyme labels or biotin anti-biotin systems, such as that described in pending U.S. patent application Serial Nos. 608,849; 070,647; 418,981; and 687,785.
  • the HGBV nucleic acid sequences may be used to gain further information on the sequence of the HGBV genome and for identification and isolation of the HGBV agent.
  • this knowledge will aid in the characterization of HGBV including the nature of the HGBV genome, the structure of the viral particle and the nature of the antigens of which it is composed.
  • This information can lead to additional polynucleotide probes, polypeptides derived from the HGBV genome, and antibodies directed against HGBV epitopes which would be useful for the diagnosis and/or treatment of HGBV caused non- A, non-B, non-C, non-D and non-E hepatitis.
  • Synthetic oiigonucleotides may be prepared using an automated oligonucleotide synthesizer such as that described by Warner, DNA 3:401 (1984). If desired, the synthetic strands may be labeled with 32p by treatment with polynucleotide kinase in the presence of ----'P-ATP, using standard conditions for the reaction. DNA sequences including those isolated from genomic or cDNA libraries, may be modified by known methods which include site directed mutagenesis as described by Zoller, Nucleic Acids Res. 10:6487 (1982).
  • the DNA to be modified is packaged into phage as a single stranded sequence, and converted to a double stranded DNA with DNA polymerase using, as a primer, a synthetic oligonucleotide complementary to the portion of the DNA to be modified, and having the desired modification included in its own sequence.
  • Culture of the transformed bacteria, which contain replications of each strand of the phage are plated in agar to obtain plaques. Theoretically, 50% of the new plaques contain phage having the mutated sequence, and the remaining 50% have the original sequence.
  • Replicates of the plaques are hybridized to labeled synthetic probe at temperatures and conditions suitable for hybridization with the conect strand, but not with the unmodified sequence. The sequences which have been identified by hybridization are recovered and cloned.
  • PCR Polymerase chain reaction
  • LCR ligase chain reaction
  • LCR is an altemate mechanism for target amplification.
  • two sense (first and second) probes and two antisense (third and fourth) probes are employed in excess over the target.
  • the first probe hybridizes to a first segment of the target strand and the second probe hybridizes to a second segment of the target strand, the first and second segments being positioned so that the primary probes can be ligated into a fused product.
  • a third (secondary) probe can hybridize to a portion of the first probe and a fourth (secondary) probe can hybridize to a portion of the second probe in a similar ligatable fashion. If the target is initially double stranded, the secondary probes will also hybridize to the target complement in the first instance.
  • Oiigonucleotides are provided which are useful for the detection of HGB V- C. These oiigonucleotides detect isolates of HGBV-C but do not detect isolates of HGBV-A or HGBV-B. These primers are designated as SEQUENCE ID NO. 51, SEQUENCE ID NOS 53-57 and SEQUENCE ID NO 87. Other primers, designated as SEQUENCE ID NO. 27 through SEQUENCE ID NO 35, are useful for classifying genotypes of HGBV-C. As a result of studying nucleotide sequences of HGBV-C isolates, it has been found that these isolates can be divided into at least four genotypes based upon the nucleotide sequences located near the 5' end of the genome.
  • the primers are useful in amplification procedures described previously herein such as PCR.
  • Other primers which can differentiate between the genotypes can be used in PCR, while other primers are useful in GAP LCR. These are described in the following examples. These primers thus provide a method of detecting HGBV-C genotypes.
  • HGBV-C isolates obtained from 39 individuals into four genotypes. These genotypes exhibited a maximum sequence divergence of 17.4%.
  • HGBV-C oiigonucleotides described herein are useful in detecting HGBV-C nucleic acids in a test sample.
  • the genotyping of HGBV-C isolates also will aid in prognostic studies as well as in prevention and treatment of the disease caused by HGBV-C in humans.
  • the present invention will now be described by way of examples, which are meant to illustrate, but not to limit, the spirit and scope of the invention.
  • HGB V-C-specific ELISAs As described by us previously in U.S. Serial No. 08/473,475 (previously inco ⁇ orated herein by reference), the generation of HGB V-C-specific ELISAs has allowed the identification of immunopositive sera in each of several categories of human populations, including intravenous drug users, residents of West Africa, volunteer blood donors and individuals with or at risk for non- A-E hepatitis. Sera from these seropositive individuals were tested for HGBV-C viremia by the RT- PCR assays described briefly as follows, and several serum samples were found to be positive for HGBV-C viral RNA. RT-PCR was performed using degenerative NS3 oligonucleotide primers (SEQUENCE ID NOS.
  • thermocycling protocol designed to amplify specific products with oligonucleotide primers that may contain base pair mismatches with the template to be amplified (Roux, Bio/Techniques 16:812-814 [1994]). Specifically, reactions were thermocycled 43 times (94°C, 20 sec; 55°C decreasing 0.3°C/cycle, 30 sec; 72°C, 1 min) followed by 10 cycles (94°C, 20 sec; 40°C, 30 sec; 72°C, 1 min) with a final extension at 72°C for 10 minutes.
  • PCR products were separated by agarose gel electrophoresis, visualized by UV i ⁇ adiation after direct staining of the nucleic acid with ethidium bromide, then hybridized to a radiolabeled probe for GB-C (SEQUENCE ID NO 26, from position 4245 to 4432) after Southern transfer to Hybond-N+ nylon filter (available from Amersham Life Sciences, Arlington Heights, IL).
  • GB-C SEQUENCE ID NO 26, from position 4245 to 4432
  • Hybond-N+ nylon filter available from Amersham Life Sciences, Arlington Heights, IL.
  • Testing by RT- PCR of additional seropositive individuals from each of the populations listed above demonstrated a correlation between antibody presence and detection of viral RNA.
  • the PCR amplified products from 26 of these individuals were cloned into the vector pT7Blue and sequenced, following methods as described in the art.
  • oligonucleotide primers SEQ ID NO. 51 (ntrC-Sl) and SEQUENCE ID NO 52 (G131-E1 wb2), located near the 5'-terminus of the HGBV-C genome and near the N-terminus of the putative El gene, respectively, were utilized in the thermocycling protocol known in the art and previously described hereinabove, on serum-derived cDNA products generated as known in the art.
  • other oligonucleotide primers i.e., ntrC-S2
  • PCR products (SEQUENCE ID NOS 2 through 6, SEQUENCE ID NO 9 through 11, SEQUENCE ID NO 13 through 24 and SEQUENCE ID NO 60 through 86) (TABLE 1) were obtained from 39 individuals previously shown to be HGBV-C RNA positive: Four of these isolates were from individuals classified as indeterminate for the presence of antibodies to hepatitis C virus proteins, 21 of these isolates were from individuals from a region of West Africa where infection with other hepatitis viruses is endemic (this includes the co ⁇ esponding sequence from the HGBV-C genome, SEQUENCE ID NO 26, in TABLE 1, hereinbelow), four of these isolates were from non A-E hepatitis patients, five of these isolates were from patients diagnosed with aplastic anemia, six of these isolates were from multiply transfused individuals, three of these isolates were from normal blood donors from the U.S., one of these isolates was from an intravenous drug user (IVDU) and two of these isolates were from two individuals from southeast Asia
  • IVDU intrave
  • HGBN-C isolates obtained are listed in TABLE 1, including a description of the individual from which the isolates were obtained. In some cases multiple isolates obtained from a single source were sequence analyzed; specifically: SEQUENCE ID NOS 16, 20 and 84 were obtained from an individual diagnosed with aplastic anemia; SEQUENCE ID NOS 21 and 22 were obtained from a multiply transfused individual; SEQUENCE ID NOS 67 and 68, 69 and 70, 75 and 76 were obtained from three individuals from West Africa, respectively.
  • FIGURE 1 A- FIGURE IF Wisconsin Sequence Analysis Package (Version 8) and is shown in FIGURE 1 A- FIGURE IF.
  • the consensus nucleotide at each position in the alignment was determined by the base that occurred most frequently at that position.
  • the consensus line does not necessarily represent the consensus sequence of the "prototype" GB virus C isolate.
  • the dashes (-) in FIGURE 1 A- 1F represent bases identical to that shown on the consensus line which is indicated as “cons” in this FIGURE. Base deletions are indicated by periods (.) in this FIGURE.
  • bases are shown only at those positions in the alignment that differ from the consensus.
  • the sequence of the PCR primers used to amplify the isolates are not shown the alignment of this FIGURE.
  • genotypes 1 and 4 are significantly distant from each other and the other two groups, i.e., genotypes 2 and 3.
  • genotypes 1 and 4 are significantly distant from each other and the other two groups, i.e., genotypes 2 and 3.
  • Genotype 1 isolates of HGBV-C have been found exclusively among individuals from West Africa and have included the original HGBV-C isolate.
  • Sequences belonging to Genotypes 2 and 3 isolates have not demonstrated, to date, a specific geographic distribution. Complete clinical information regarding disease status or treatment regimes has not been available for all of these individuals from which sequences were obtained.
  • PCR primers which were utilized to amplify sequences from the NS3 region and the 5 '-terminal portion (GBV-C [SEQUENCE ID NO 51] - Elwb2 [SEQUENCE ID NO 52]) of the genome were located in regions that were not well conserved in all isolates, not all HGBV-C viremic samples tested may have been detected by the RT-PCR assays employed here. It was hypothesized that utilization of PCR primers from a highly conserved region of the HGBV-C genome, as have been found in the HCV 5' untranslated region [Cha, et al. J. Clin. Microbiol. 29:2528-2534 (1991)], should allow more accurate detection of HGBV-C viremic samples.
  • the primers ntrC-S 1/ntrC-al (SEQUENCE ID NO 51/SEQUENCE ID NO 54) and ntrC-S2/ntrC-a2 (SEQUENCE ID NO 53/SEQUENCE ID NO 55) were used in independent PCRs or in combination in a nested PCR experiment.
  • the primers ntrC-3F/ntrC-4R (SEQUENCE ID NO 56/SEQUENCE ID NO 57) were used in combination in separate PCRs.
  • the first round amplification was performed on serum cDNA products generated as described earlier, using 2 mM MgCl2 and 1 ⁇ M primers (both sense and antisense), as follows. Reactions were subjected to 35-40 cycles of denaturation-annealing-extension (94°C, 20 sec; 55°C, 30 sec; 72°C, 45 sec) followed by a 10 min extension at 72°C. Completed reactions were held at 4°C. A second round of amplification performed as either a fully nested or a hemi-nested reaction, if necessary, was performed utilizing 2 mM MgCl2, 1 ⁇ M sense and antisense primers and 4% of the first PCR products as template.
  • the second round of amplification employed a thermocycling protocol identical that utilized in the first round of PCR.
  • PCR products were separated by agarose gel electrophoresis and visualized by UV irradiation after direct staining of the nucleic acid with ethidium bromide.
  • UV irradiation after direct staining of the nucleic acid with ethidium bromide.
  • the products of a single round of PCR amplification were transfened to Hybond-N+ nylon filter and then hybridized to a radiolabeled probe for HGBV-C.
  • Results obtained from these experiments confirmed the presence of HGBV-C RNA in 38 out of 39 individuals whose sera had previously tested positive for HGBV-C RNA by using the helicase region primers as stated hereinabove.
  • RT-PCR experiments were conducted in which various sense primers (ntrC-Sl [SEQUENCE ID NO 51], ntrC-3F [SEQUENCE ID NO 56), ntrC-Al [SEQUENCE ID NO 54], ntrC-A2 [SEQUENCE ID NO 55], ntrC-4R [SEQUENCE ID NO 57], ntrC-5R [SEQUENCE ID NO 87]).
  • thermocycling protocol designed to amplify DNA sequences that may contain base pair mismatches between the template and the primer(s) as described by Roux, BioTechniques 16:812-814 (1994)]. Specifically, reactions were thermocycled 43 times (94°C, 20 sec; 55°C decreasing 0.3°C/cycle, 30 sec; 72°C, 1 min) followed by 10 cycles (94°C, 20 sec; 40°C, 30 sec; 72°C, 1 min) with a final extension at 72°C for 10 minutes.
  • PCR products were separated by agarose gel electrophoresis and visualized by UV inadiation after direct staining of the nucleic acid with ethidium bromide.
  • the nucleic acids then were transfe ⁇ ed by Hybond-N+ nylon filters (available from Amersham Life Sciences, Arlington Heights, EL) and then hybridized to the appropriate radiolabeled probe for HGB V- C (SEQUENCE ID NO 26, positions 13 to 631 ).
  • NS3-derived HGBV-C degenerate primers (SEQUENCE ID NO 88 and SEQUENCE ID NO 89) detected RNA in 7/12 specimens as determined by ethidium bromide staining and 8/12 specimens by southern analysis. These data indicate that some HGBV-C 5'-end primers pairs may be more sensitive than others for detecting HGBV-C viremia, even though all 5'-end primers are derived from regions exhibiting a high degree of nucleotide sequence conservation among all HGBV-C isolates.
  • HGBV-C NS3 helicase derived PCR primers may not be as sensitive as the primers derived from the 5' end of the genome for the detection of HGBV-C nucleic acids.
  • TABLES 2A and 2B indicate primer pairs wherein Sl conesponds to SEQUENCE ID NO 51, S2 co ⁇ esponds to SEQUENCE ID NO 53, 5R conesponds to SEQUENCE ID NO 87, 3F co ⁇ esponds to SEQUENCE ID NO 56, Al conesponds to SEQUENCE ID NO 54, A2 co ⁇ esponds to SEQUENCE ID NO 55, 4R co ⁇ esponds to SEQUENCE ID NO 57 and NS3 co ⁇ esponds to SEQUENCE ID NO 88 and 89.
  • HGBV-C-la-sl SEQUENCE ID NO 28
  • HGBV-C-la-s2 SEQUENCE ID NO 32
  • HGBV-C- la-al SEQUENCE ID NO 27
  • HGBV-C-la-a2 SEQUENCE ID NO 31
  • HGBV-C-lbc-sl SEQUENCE ID NO 29
  • HGBV-C-lbc-s2 SEQUENCE ID NO 33
  • HGBV-C-lbc-al SEQUENCE ID NO 30
  • HGBV-C-lbc-a2 SEQUENCE ID NO 34
  • Primers listed above with a "la” designation are selective for HGBV-C genotype 1; primers with a “lb” designation are selective for HGBV-C genotype 2; primers with a “lc” designation are selective for HGBV-C genotype 3.
  • the genotype of the particular HGBV-C isolate is determined by the presence or absence of a PCR amplification product of the predicted size as visualized by agarose gel electrophoresis and ethidium bromide staining.
  • genotypes can be identified by determining the nucleic acid sequence of the PCR amplification product produced using one of the HGBV-C primer pairs. The determined sequence is then compared with the sequence from the homologous region of other HGBV-C isolates. This is accomplished by sequence alignment and subsequent phylogenetic analysis using methods known in the art.
  • Example 3 GAP LCR Detection of HGBV-C Genotypes
  • oligonucleotide primers that can be used in a GAP-LCR assay to distinguish between members of HGBV-C genotypes 1, 2 or 3.
  • double-gap LCR is performed as follows and as detailed in U.S. Patent No. 5, 427,930, previously inco ⁇ orated herein by reference.
  • Double gaps are represented herein as "DG p, q" where in “p” is the number of bases in the gap of one strand, and “q” is the number of bases in the gap of the other strand.
  • DG2,2 the number of bases in the ligation
  • Double gap LCR is performed for 30-50 cycles consisting of a 65 second incubation at 85°C and a 65 second incubation at 50°C.
  • the oiigonucleotides used are presented hereinbelow as SEQUENCE ID NOS. 35 through 50, and are specific for the 5' end of HGBV-C.
  • Reactions are run in a buffer containing 50 mM EPPS pH 7.8, 100 mM KCI, 10 mM MgCl2, 1 mM DTT, 10 mM NH4CI, 100 ⁇ m NAD, 10 ⁇ g ml BSA, 5 x 10 1 1 each oligonucleotide listed hereinabove, 1 ⁇ m 2' -deoxyguanosine 5'triphosphate, 0.5 units Thermus DNA polymerase (Molecular Biology Resources, Inc., "MBR"), and 3400 units Thermus thermohilus DNA ligase. Reaction volume is 50 ⁇ l and each reaction is overlaid with 25 ⁇ l of mineral oil prior to cycling. Following amplification, reactions are diluted 1:1 with IM X ® diluent buffer
  • the LCR amplification products are detected via a sandwich immunoassay performed using the Abbott IM ⁇ ® automated immunoassay system.
  • RNA viruses such as picornaviruses and pestiviruses possess large 5' nontranslated regions (NTRs). These large NTRs control the initiation of cap-independent translation by functioning as intemal ribosome entry sites (IRESs) (Pelletier and Sonenberg, Nature (London) 334:320- 325).
  • IRES intemal ribosome entry sites
  • the IRES is thought to form a specific RNA structure which allows ribosomes to enter and begin translation of an RNA without using the cellular machinery required for cap-dependent translation initiation.
  • the large 5' NTR of HCV has been shown to possess an IRES (Wang et al. J. Virol. 67:3338-3344, 1993). Due to the high level of sequence conservation between the 5' NTRs of GBV-B and HCV, it was reasoned that GBV-B may also contain an IRES.
  • the 5' NTR of this virus was used to replace the 5' NTR of hepatitis A virus (HAV) in the pLUC-HAV-CAT plasmid described by Whetter et al. ( J. Virol. 68:5253-5263, 1994).
  • the 5' NTR of GBV- B was amplified from a plasmid clone using SEQUENCE ID NO. 58 (UTR-B.l) and SEQUENCE ID. NO.
  • NTR-B-al primers
  • a 50 ⁇ l PCR was set up using a Perkin-Elmer PCR kit as described by the manufacturer with 1 ⁇ M primers, 2 mM MgCl2 and approximately 10 ng of plasmid. This reaction was amplified for 20 cycles (94°C, 20 sec; 55°C, 30 sec; 72°C, 30 sec) followed by a final extension at 72°C for 10 min. The completed reaction then was held at 4°C. This product was extracted with phenol hloroform and precipitated as described in the art.
  • the 3' terminal adenosine residues added by the AmpliTaq® polymerase were removed from this product by incubation with T4 DNA polymerase and deoxynucleotide triphosphates as described (Sambrook et al, Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, 1989). After heat inactivation, the product was digested with Xba I and gel purified as described in the art. The purified product was ligated to pHAV-CATl (Whetter et al. J. Virol.
  • the 1.3 kbp product from these reactions was gel purified and cloned into pLUC- HAV-CAT (Whetter et al. J. Virol. 68:5253-5263, 1994) that had been digested with HindlH, end-filled with Klenow polymerase and deoxynucleotide triphosphates, heat-inactivated, digested with Not I, treated with bacterial alkaline phosphatase, extracted with phenolxhloroform, and precipitated as described in the art.
  • the resultant plasmid, pLUC-GBB-CAT was used in in vitro transcription-translation experiments to test for an IRES function.
  • GBV-B's 5'NTR contains an IRES. Further studies of these plasmids, both in vitro and in vivo are ongoing to better characterize the IRES in GBV-B.
  • NAME Porembski, Priscilla E.
  • MOLECULE TYPE DNA (genomic)
  • GAGACCGTTA TCCCTCTGGG CAAACGACGC TCACGTACGG TCCACGTCGC CCTTCAATGC 300
  • TTTTCTTCCT ATACCGATCA TGGCAGTCCT TCTGCTTCTA CTCGTTGTGG AGGCCGGGGC 540
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • CCGAGCCCGT CATCCGCCTG GGCTAACGAC GCCCACGTAC GGTCCACGTC GCCCTTCAAT 300
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic) (xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 19 :
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic) (xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 24 :
  • TCCCCCCCCT TAACA ⁇ CTGC ⁇ TGCTTCTCG GC ⁇ CTG ⁇ GGT GTC ⁇ GAGGT ⁇ TTGGGTGGGG 1 80
  • CTAGGGGCCT GTTGTGGCAT CCAGGACTCC GGCTTCCTCC CCCTGAGATT GCTGGTATCC 8940
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • SEQUENCE DESCRIPTION SEQ ID NO:31: TTTCCCTCCA TAAGCGCG 18
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Virology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Communicable Diseases (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Analytical Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des acides nucléiques du virus de l'hépatite C du type GB et notamment des sondes et des amorces oligonucléotidiques, utiles dans la détection de génotypes du virus de l'hépatite C du type GB.
EP96930516A 1995-08-14 1996-08-14 Detection de genotypes du virus gb de l'hepatite Withdrawn EP0845049A2 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US226595P 1995-08-14 1995-08-14
US580038 1995-12-21
US08/580,038 US5807670A (en) 1995-08-14 1995-12-21 Detection of hepatitis GB virus genotypes
PCT/US1996/013171 WO1997008531A2 (fr) 1995-08-14 1996-08-14 Detection de genotypes du virus gb de l'hepatite
US2265 1997-12-31

Publications (1)

Publication Number Publication Date
EP0845049A2 true EP0845049A2 (fr) 1998-06-03

Family

ID=26670161

Family Applications (1)

Application Number Title Priority Date Filing Date
EP96930516A Withdrawn EP0845049A2 (fr) 1995-08-14 1996-08-14 Detection de genotypes du virus gb de l'hepatite

Country Status (5)

Country Link
US (1) US5807670A (fr)
EP (1) EP0845049A2 (fr)
JP (1) JPH11511027A (fr)
CA (1) CA2228820A1 (fr)
WO (1) WO1997008531A2 (fr)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5981172A (en) * 1994-02-14 1999-11-09 Abbott Laboratories Non-A, non-B, non-C, non-D, non-E Hepatitis reagents and methods for their use
IT1283893B1 (it) * 1996-01-24 1998-05-07 Sorin Biomedica Diagnostics Sp Metodo per rilevare sequenze nucleotidiche di virus associati a epatiti nona-none, peptidi e composizioni
US6436638B1 (en) * 1996-05-09 2002-08-20 Metropolitan Water District Of Southern California Cryptosporidium detection method
US7244585B1 (en) 1999-06-04 2007-07-17 The Board Of Regents Of The University Of Texas System 3′ Sequence of the GB virus B (GBV-B) genome
US7141405B2 (en) * 1999-06-04 2006-11-28 Board Of Regents, The University Of Texas System Chimeric GB virus B (GBV-B)
US20060105365A1 (en) * 2004-09-27 2006-05-18 Annette Martin Chimeric GB virus B (GBV-B)

Family Cites Families (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4743535A (en) * 1984-11-07 1988-05-10 Miles Inc. Hybridization assay employing labeled probe and anti-hybrid
US4876187A (en) * 1985-12-05 1989-10-24 Meiogenics, Inc. Nucleic acid compositions with scissile linkage useful for detecting nucleic acid sequences
CN1074422C (zh) * 1987-11-18 2001-11-07 希龙股份有限公司 制备含有hcv表位的分离多肽的方法
WO1990000597A1 (fr) * 1988-07-06 1990-01-25 Genelabs Incorporated Virus et antigenes d'hepatite serique non a et n b
JP2696116B2 (ja) * 1988-09-30 1998-01-14 財団法人阪大微生物病研究会 非ａ非ｂ肝炎患者の血清と抗原抗体反応するペプチド、及び該ペプチドをコードするｄｎａ
US5399346A (en) * 1989-06-14 1995-03-21 The United States Of America As Represented By The Department Of Health And Human Services Gene therapy
EP0529493B1 (fr) * 1991-08-27 1997-12-17 F. Hoffmann-La Roche Ag Procédés et réactifs pour la détection de l'hépatite C
US5576302A (en) * 1991-10-15 1996-11-19 Isis Pharmaceuticals, Inc. Oligonucleotides for modulating hepatitis C virus having phosphorothioate linkages of high chiral purity
WO1994018217A1 (fr) * 1993-02-03 1994-08-18 Abbott Laboratories Reactifs des hepatites non-a, non-b, non-c, non-d, non-e et leurs procedes d'utilisation
CA2145290C (fr) * 1992-09-28 2002-03-05 Jang H. Han Methodes et compositions pour la regulation de la traduction des proteines du vhc
ES2113673T3 (es) * 1993-08-24 1998-05-01 Akzo Nobel Nv Lentes oftalmicas.
CA2166313A1 (fr) * 1994-02-14 1995-08-17 John N. Simons Reactifs pour l'hepatite non-a, non-b, non-c, non-d et procede pour leur utilisation
US5981172A (en) * 1994-02-14 1999-11-09 Abbott Laboratories Non-A, non-B, non-C, non-D, non-E Hepatitis reagents and methods for their use
JPH10503642A (ja) * 1994-05-20 1998-04-07 ジェネラブス テクノロジーズ， インコーポレイテッド Ｇ型肝炎ウイルスおよびその分子クローニング
AU2549195A (en) * 1994-05-20 1995-12-18 Genelabs Technologies, Inc. Non-A/non-B/non-C/non-D/non-E hepatitis agents and molecular cloning thereof
AU2594195A (en) * 1994-05-20 1995-12-18 Genelabs Technologies, Inc. Detection of viral antigens coded by reverse-reading frames
US6370502B1 (en) * 1999-05-27 2002-04-09 America Online, Inc. Method and system for reduction of quantization-induced block-discontinuities and general purpose audio codec
EP1768150B1 (fr) * 2005-09-26 2010-02-17 ABB Technology AG Disjoncteur à haute tension avec pouvoir de coupure ameliorée
KR101252635B1 (ko) * 2006-04-20 2013-04-10 (주)아모레퍼시픽 리파아제 저해제 및 친유성 오일흡수제를 포함하는 약학조성물 및 이로부터 제조된 경구 투여용 제제
AU2008356409B2 (en) * 2008-05-13 2012-01-19 Astrazeneca Ab Quinuclidine derivatives as muscarinic M3 receptor antagonists
US8344271B1 (en) * 2009-08-24 2013-01-01 Falk Jr David C Luggage having a built-in scale configured to slide into and out of the luggage base, where the scale can measure weight in either configuration
US8357509B2 (en) * 2009-12-17 2013-01-22 Auburn University Microbial expression of tobacco osmotin for biocidal and medical applications

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9708531A3 *

Also Published As

Publication number Publication date
US5807670A (en) 1998-09-15
JPH11511027A (ja) 1999-09-28
CA2228820A1 (fr) 1997-03-06
WO1997008531A3 (fr) 1997-04-17
WO1997008531A2 (fr) 1997-03-06

Similar Documents

Publication Publication Date Title
JP3701754B2 (ja) Ｎａｎｂｖの診断法：ｃ型肝炎ウイルスのスクリーニングに有用なポリヌクレオチド
EP0856051B1 (fr) Nouvelle sequence terminale 3' du genome du virus de l'hepatite c et son utilisation diagnostique et therapeutique
EP0585398B1 (fr) Sequences genomiques du virus de l'hepatite c utilisees en diagnostic et en therapeutique
Turkoglu et al. Detection of hepatitis E virus RNA in stools and serum by reverse transcription-PCR
LV10726B (en) Hepatitis c diagnostics and vaccines
WO1997007224A1 (fr) Reactifs et procedes permettant de moduler la traduction de proteines de l'hepatite gbv
JP2005040145A (ja) Ｈｃｖ関連オリゴヌクレオチドおよびｈｃｖ単離物の特定領域遺伝子の増幅方法
WO2000046407A2 (fr) Procedes relatifs a l'utilisation du virus tt
WO1994018217A1 (fr) Reactifs des hepatites non-a, non-b, non-c, non-d, non-e et leurs procedes d'utilisation
Barrera et al. Analysis of host range phenotypes of primate hepadnaviruses by in vitro infections of hepatitis D virus pseudotypes
WO1995021922A2 (fr) Reactifs pour l'hepatite non-a, non-b, non-c, non-d et procede pour leur utilisation
US7455969B2 (en) Highly permissive cell lines for hepatitis C virus RNA replication
US20060228702A1 (en) Polynucleotide probe and primer derived from hepatitis E virus recovered from japanese, chip including the same, kit including the same, and method of detecting hepatitis E virus genome using the same
WO1997008531A2 (fr) Detection de genotypes du virus gb de l'hepatite
WO1997008531A9 (fr) Detection de genotypes du virus gb de l'hepatite
US5981172A (en) Non-A, non-B, non-C, non-D, non-E Hepatitis reagents and methods for their use
US5552310A (en) Replication of hepatitis C virus genome and identification of virus having high infectivity
US5709997A (en) Nucleic acid detection of hepatitis GB virus
van Doom et al. Rapid detection of hepatitis C virus RNA by direct capture from blood
JPWO2005028652A1 (ja) 新規ｈｃｖ株由来の核酸、遺伝子、及び該遺伝子を利用したレプリコン複製細胞
EP1036847B1 (fr) Amorces oligonucléotidiques pour une transcription inverse efficace de l'ARN du virus de l'Hépatite C et ses procédés d'utilisation
Aaronson et al. Viral genes involved in leukemogenesis. I. Generation of recombinants between oncogenic and nononcogenic mouse type-C viruses in tissue culture.
US20040229213A1 (en) Exhaustive analysis of viral protein interactions by two-hybrid screens and selection of correctly folded viral interacting polypeptides
Stuyver et al. The use of a Line Probe Assay as a tool to detect new types or subtypes of the hepatitis C virus
US20020102534A1 (en) Exhaustive analysis of viral protein interactions by two-hybrid screens and selection of correctly folded viral interacting polypeptides

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19980213

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH DE ES FR GB IT LI NL

17Q First examination report despatched

Effective date: 20010129

GRAH Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOS IGRA

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20031001